Hematology and serum biochemistry reference ranges of healthy captive Tasmanian devils (Sarcophilus harrisii) and their association with age, gender and seasonal variation

Accepted Manuscript Title: Hematology and serum biochemistry reference ranges of healthy captive Tasmanian devils (Sarcophilus harrisii) and their association with age, gender and seasonal variation Author: Hayley J. Stannard Paul Thompson Bronwyn M. McAllan David Raubenheimer PII: DOI: Reference:

S1616-5047(16)30022-2 http://dx.doi.org/doi:10.1016/j.mambio.2016.03.007 MAMBIO 40821

To appear in: Received date: Accepted date:

19-11-2015 29-3-2016

Please cite this article as: Stannard, Hayley J., Thompson, Paul, McAllan, Bronwyn M., Raubenheimer, David, Hematology and serum biochemistry reference ranges of healthy captive Tasmanian devils (Sarcophilus harrisii) and their association with age, gender and seasonal variation.Mammalian Biology http://dx.doi.org/10.1016/j.mambio.2016.03.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Hematology and serum biochemistry reference ranges of healthy captive Tasmanian devils

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(Sarcophilus harrisii) and their association with age, gender and seasonal variation

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Hayley J. Stannard1, Paul Thompson2, Bronwyn M. McAllan3 and David Raubenheimer1,4

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Centre, University of Sydney, Sydney NSW 2006, Australia

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Taronga Wildlife Hospital, Mosman, NSW 2088, Australia

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Discipline of Physiology, and Bosch Institute, School of Medical Sciences, the University of Sydney,

School of Biological Sciences, University of Sydney, Sydney NSW 2006, Australia and Charles Perkins

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New South Wales 2006, Australia

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Faculty of Veterinary Science, University of Sydney, Sydney NSW 2006, Australia

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Correspondence:

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Hayley Stannard

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School of Biological Sciences and Charles Perkins Centre,

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University of Sydney,

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Level 4, East Wing (L4E75)

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Charles Perkins Centre D17

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Sydney NSW 2006, Australia

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P: +61 2 8627 0638

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E: [email protected]

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Short title: Hematology and blood chemistry of Tasmanian devils

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Abstract

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The Tasmanian devil (Sarcophilus harrisii) is the largest extant carnivorous marsupial. The Tasmanian

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devil is currently listed as endangered and is under threat from a contagious cancer. The aims of the study

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were to determine hematology and blood chemistry reference intervals for captive Tasmanian devils and

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determine the influence of three biological factors on blood variables. Hematology and blood chemistry

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data were analyzed retrospectively from medical reports obtained from Taronga Zoo. Samples were

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analyzed using current technology at the time of collection. Thirty seven variables were analyzed for 104

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blood samples from 1992 until 2015. Data were statistically analyzed for differences between age, gender

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and season. Generally Tasmanian devils have higher serum concentrations of albumin (ALB) and

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creatinine and lower alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline

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phosphatase (ALP) and amylase (AMY) compared with other dasyurids. Younger animals tended to have

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significantly higher serum concentrations of ALP, AST and phosphorus, while total protein and globulin

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activity in younger animals was less than in older animals. Hemoglobin, total protein and AST

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concentrations were influenced by season, with higher concentrations observed in either spring or

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summer. Lymphocyte, and erythrocyte counts, and serum concentrations of lipase and AMY were

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significantly higher in females compared with males. The reference ranges determined here can be used in

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the health assessment of captive Tasmanian devils and for those used in translocation programs in the

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future.

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Key words Blood chemistry; captive management; dasyurid; health assessment; marsupial

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Introduction

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The Tasmanian devil (Sarcophilus harrisii) is the largest extant carnivorous marsupial, ranging up to

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10kg body weight (Jones, 2008). Devils eat medium sized mammals, fish and birds (Jones, 2008;

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Pemberton et al., 2008). In the wild, the geographical range of the devil is currently restricted to the island

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state of Tasmania; however they were once more widespread across mainland Australia. In the past

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Tasmanian devils were persecuted for killing livestock and now they are under threat from a contagious

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cancer (Hawkins et al., 2006; McCallum et al. 2007). The Tasmanian devil population has declined by >

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60% and is listed as ‘Endangered’ on the IUCN Red List (Hawkins et al. 2008).

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Current data available on hematology and blood chemistry of Tasmanian devils is limited, with

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gaps in the variables reported and small samples sizes (Bartels et al., 1966; Parsons et al., 1970; Nicol,

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1982; Clark, 2004; Holz, 2008). Recently data have become available for wild devils (Peck et al. 2015).

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However, thus far these studies have shown that Tasmanian devils have notably high serum

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concentrations of acid phosphatase, phosphorous and urea (Parsons et al., 1970). Devils also have a

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significant proportion of alkali resistant hemoglobin compared with eutherian species (Parsons et al.,

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1970). Preliminary investigation of a lactating female shows they have lower calcium and alkaline

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phosphatase (ALP) compared to males, but in that study only one lactating female and two males were

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sampled (Parsons et al., 1970). Devils generally conform to the typical marsupial serum chemistry having

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high blood glucose and urea when compared to humans, but specific ranges are species dependent

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(Parsons et al., 1971).

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Abnormal hematology and blood chemistry values can indicate poor health and disease affecting

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an individual. Baseline reference data is required for determining health and illness in individuals. At this

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stage suitable reference data is not available for captive Tasmanian devils, although there are reference

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intervals available for wild Tasmanian devils and other dasyurids (for example, Schmitt et al., 1989;

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Bradley, 1990; Haynes and Skidmore, 1991; Clark, 2004; Stannard et al., 2013; Peck et al., 2015). Blood

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variables can be influenced by age, season, gender, nutrition, and illness/disease, with the effects of these

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factors on blood variables varying from species to species (for example, Schmitt et al., 1989; Haynes and

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Skidmore, 1991; Bradley, 1990; Wells et al., 2000; Hall et al., 2007). Large populations of devils are

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being kept in captivity as part of the insurance population and this provides an opportunity to collect and

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develop baseline data from these animals as a preliminary step in monitoring translocated or reintroduced

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animals and can be used as a comparison for wild individuals.

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The present study involved compiling hematology and blood chemistry data available for healthy

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captive Tasmanian devils from Taronga Zoo, with the aim of developing baseline reference ranges.

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Comparisons were made between gender, season and age to determine how these factors influenced the

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blood variables studied.

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Materials and Methods

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Animals and blood sample analysis

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Records were collected from Taronga Zoo Medical Reports on their Tasmanian devils from 1992 to 2015

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from captive-bred animals. Blood samples were taken as part of routine health checks, during quarantine

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or as part of an enquiry into clinical signs of illness. Results from clinically ill animals (based on

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veterinarian notes) were not included in the analysis. During blood collection animals were sedated with

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Isoflurane (IsoFlo, Abbott Australasia Pty Ltd., Botany NSW) and blood was collected from the

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saphenous, jugular, cephalic or tail vein. The blood collected was split between a serum separator tube for

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clinical chemistry (Becton Dickinson, North Ryde, NSW) and an ethylenediamineteraacetic (EDTA) tube

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for hematology analysis (Becton Dickinson, North Ryde, NSW). Tasmanian devils were maintained in

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outdoor enclosures and exposed to natural light cycles and temperatures at either Taronga Zoo (Mosman,

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NSW) or Taronga Western Plains Zoo (Dubbo, NSW). All devils were maintained on a carnivorous diet

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that included whole quail, chicken wings, kangaroo pieces, and gutted rabbit carcass.

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Blood variables were measured with a Reflotron Instrument (Roche Diagnostics Ltd.,

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Switzerland) and an IDEXX VetTest Chemistry Analyzer (Idexx Laboratories, Rydalmere) from 1992 till

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2010. After November 2010 blood was analyzed in a VetScan (VS2) Chemical Analyzer (Abaxis, CA,

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USA). Taronga Western Plains Zoo samples were sent to Pathology West (Dubbo, NSW) for analysis.

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The hematology variables measured were erythrocyte count, mean corpuscular volume (MCV), mean

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corpuscular hemoglobin (MCV), mean corpuscular hemoglobin concentration (MCHC), hemoglobin

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(HGB), hematocrit (HCT), total leucocyte count (WBC) and WBC differential using a IDEXX

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VetAutoread (Idexx Laboratories, Rydalmere). Biochemistry variables analyzed included: anion gap,

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concentrations of total lipase, gamma-glutamyl transferase (GGT) , cholesterol, chloride, total carbon

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dioxide (Total CO2), creatine kinase (CK), glucose, urea nitrogen (BUN), creatinine, calcium,

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phosphorus, sodium, potassium, magnesium total protein, albumin (ALB), globulin, alanine

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aminotransferase (ALT), total bilirubin, amylase (AMY), alkaline phosphatase (ALP), aspartate

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aminotransferase (AST).

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Data analysis

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Reference Value Advisor v.2.1 was used to determine reference intervals and 90% confidence intervals

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for hematology and biochemistry (Geffre et al., 2011). For all variables where >40 samples were

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available non-parametric test data were used to determine reference and confidence intervals from the

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Reference Value Advisor program, and box-cox transformed data was used for variables with ≤40

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samples. The software tested normality using Anderson-Darling and Q-Q plots, and outliers using Dixon

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Reed and Tukeys’ tests (Geffre et al., 2011). Outliers were removed prior to further statistical analysis.

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ANOVAs and Tukeys’ posthoc tests were used to determine significant differences between genders,

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ages, and seasons. Employing SPSS, a mixed-model multivariate analysis was performed on data to

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account for age, gender and season where enough data were available and controlled for repeated

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measures. Variables were grouped into season based on the southern hemisphere, for example summer:

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December - February.

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Results

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In total 104 individual Tasmanian devil blood samples were available for analysis from March 1992 to

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June 2015. The samples came from 50 Tasmanian devils (28 male and 22 female). Results for reference

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intervals, medians and confidence intervals are presented for hematology in Table 1 and those for serum

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biochemistry are in Table 2.

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Age

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WBC were significantly (F7,103= 3.9 P<0.01) higher in 1 year olds compared with 3 and 4 year olds (Table

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3). HCT was significantly (F7,101= 8.2 P<0.01) lower in 7 year olds compared to 1, 2, 3 and 4 year olds, in

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both males (F6,57= 8.3 P<0.01) and females (F6,41= 4.5 P<0.01). The youngest two age groups had lower

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neutrophils (F7,103= 7.5 P<0.01), and higher lymphocytes (F7,103= 6.9 P<0.01; Table 3) than the oldest two

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age groups.

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Age influenced globulin, total protein, ALP, AST, and P activity. Total globulins were

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significantly lower in animals under one year of age compared to the other ages (F7,91= 3.7 P<0.01; Table

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3). Globulins were significantly different in males, with animals under one year of age having

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significantly lower levels than 1, 3, 4 and 7 year olds (F4,49= 3.5 P<0.05). Some age groups had a small

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sample size and statistical significance could not be determined for all male age groups. Globulins were

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not significantly different with relation to age in females (F7,40= 1.4 P=0.24). Total protein was

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significantly lower in 7 year olds compared to 2, 3 and 4 year olds; and in under one year olds compared

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to 1, 2, 3 and 4 year olds (F7,91= 7.3; P<0.01). Animals under two years of age had significantly (F7,93=

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25.4 P<0.01) higher ALP activity than animals aged two years and older (Table 3). Animals under 1 year

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of age had significantly (F7,66= 4.2 P<0.01) higher AST concentrations than 1 and 3 year olds, and 1 year

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olds had significantly lower concentrations than 7 year old animals (Table 3). Phosphorus concentrations

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were significantly higher in animals under one year of age (F7,93= 10.6; P<0.01) in both males (F6,51=

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17.3; P<0.01) and females (F5,38= 5.5; P<0.01) compared to all other ages.

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Season

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Hemoglobin concentrations were significantly (F3,96= 3.5 P<0.05) higher in spring compared with

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summer and autumn (Table 4). When controlled for gender the males had significantly (F3,55= 4.2 P<0.05)

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higher mean HGB levels in spring (149.0 ± 19.1 g/L, n= 29) compared with autumn (130.4 ± 17.9 g/L, n=

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16). Age of the males also affected HGB (F6,54= 4.5 P<0.01) with higher mean values found in 3 and 4

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year olds (153.7 ± 15.3 g/L, n= 17) compared with 6 and 7 year olds (123.4 ± 16.1 g/L, n= 10). Total

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protein values were higher in spring and summer compared to autumn (F3,91= 4.4 P<0.05). When

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separated for gender only females had significantly higher mean protein concentrations in spring (64.4 ±

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4.6 g/L, n=14) compared to autumn (55.9 ± 6.2 g/L, n =7; F3,41= 5.0 P<0.05). Overall, AST was also

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influenced by season (F3,66= 3.5; P<0.05) with higher concentrations observed in summer compared to

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spring which was due to the male devils (F3,35= 3.7 P<0.05): summer 56.7 ± 28.7 IU/L (n= 16), autumn

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55.5 ± 19.7 IU/L (n= 8) and spring 34.3 ± 12.1 IU/L (n= 11). Samples for winter (n= 3 - 6 depending on

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parameter) were limited and no significant variables could be determined for winter values.

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Gender

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Females had significantly higher lymphocytes (x 109/L; F1,15= 4.6 P=0.05) and erythrocyte counts (x

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109/L; F1,15= 4.7 P=0.05) than males (Table 4). Gender influenced the levels of lipase and AMY present

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in the serum. Lipase was significantly (F1,5= 16.1 P<0.05) higher in females compared with males,

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however the sample size for lipase was small and further data are needed. AMY concentrations were

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significantly higher in females compared with males (F1,54= 4.8 P<0.05; Table 4).

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Discussion

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The paper presents reference intervals for hematology and serum biochemistry of captive Tasmanian

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devils. Age, gender and season were found to influence certain hematology and serum biochemistry

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variables of the devil. ALP, AST, P, total protein and globulin concentrations were influenced by age.

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Gender influenced the levels of lymphocytes, erythrocytes, lipase and AMY present in the serum with

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females having higher levels than males. Season influenced HGB, total protein and AST concentrations.

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Sample sizes for some parameters were limited, and thus statistical comparisons could not be made for all

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variables for age, season and gender. The data provides a baseline for a range of variables for captive

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Tasmanian devils and provides a basis for health assessment in this species.

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For the most part previously published data on Tasmanian devils are similar to reference intervals

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determined in our study (Bartels et al., 1966; Parsons et al., 1970; Nicol, 1982; Clark, 2004; Holz, 2008;

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Peck et al., 2015). In comparison with other dasyurids, Tasmanian devils have higher MCV levels than

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dunnarts (Sminthopsis spp.) and higher mean MCH levels than western quolls (Dasyurus geoffroii)

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(Haynes and Skidmore, 1991; Svensson et al., 1998). Eosinophils in devils were detected in low numbers.

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Low eosinophil counts were also found for quolls and were extremely rare in dunnarts, with only one

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eosinophil cell detected during analysis of numerous stripe-faced dunnart (Sminthopsis macroura) and

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brown antechinus (Antechinus stuartii) samples, and were not detected at all in samples from the fat-tailed

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dunnart (Sminthopsis crassicaudata) (Cheal et al., 1976; Melrose et al., 1987; Haynes and Skidmore,

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1991; Stannard et al., 2013). No basophils were detected in the Tasmanian devil samples which is

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consistent with findings for other dasyurid species (Parsons et al. 1970; Cheal et al. 1976; Haynes and

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Skidmore 1991; Stannard et al. 2013). Low eosinophil and basophil counts are likely due to the animals

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being in a captive environment and because they are treated for helminths and other parasites (Klion and

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Nutman, 2004).

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In the past it has been noted that Tasmanian devils have higher ALB activity than quolls and

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lower ALP and AMY activity (Parsons et al., 1970), which is consistent with the findings of this study. It

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was also found in the present study that ALT and AST activity were lower, and creatinine activity higher

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in the devils compared to values reported for western and eastern quolls (Dasyurus viverrinus), possibly

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due to differences in diet type and macronutrient composition (Svensson et al. 1998; Stannard et al.,

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2013). Liver function variables such as ALT, AST, ALB and Total bilirubin generally fall within the

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range of those determined previously for marsupials (Parsons et al., 1971, Schmitt et al., 1989; McKenzie

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et al., 2002; Barnes et al., 2008; Reiss et al., 2008; Stannard et al., 2013). GGT is lower than that

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determined for the tammar wallaby but falls within the range of that determined for the brush-tailed rock-

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wallaby (Petrogale penicillata; McKenzie et al., 2002; Barnes et al., 2008). ALT was lower than that

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determined for other dasyurids and wild Tasmanian devils but was consistent with some previously

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reported Tasmanian devil data (Parsons et al., 1970; Parsons et al., 1971; Bradley, 1990; Stannard et al.,

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2013; Peck et al., 2015).

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Higher concentration of ALP activity were seen in younger Tasmanian devils and this result was

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expected as ALP mobilization in serum is related to growth (Weiss and Wardrop, 2011), as noted

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previously in young dasyurids (Svensson et al. 1998; Stannard et al., 2013; Peck et al., 2015) and other

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mammalian species (McKenzie et al., 2002; Hall et al., 2007; Barnes et al., 2008; Reiss et al., 2008). ALP

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values were generally lower than those observed for quolls of the same age (Svensson et al., 1998;

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Stannard et al., 2013), possibly due to differences in life history traits (such as sexual maturity,

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senescence and lifespan). Growth and mobilization of bodily resources (Weiss and Wardrop, 2011) is also

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the likely reason for young Tasmanian devils having significantly higher P concentration than older

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individuals.

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Young Tasmanian devils were found to have significantly lower concentrations of total globulins

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which is consistent with wild devils and other mammals (Harvey et al., 2007; Ahlers et al., 2011; Peck et

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al., 2015). Younger animals are more likely to have lower globulin activity as their immune responses and

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antigens are still developing whereas older animals have been exposed to pathogens (occasionally

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helminths and fleas) and are experienced at mounting an immune response (Mitchell et al., 1999). The

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general trend of globulin activity in the devils is consistent with the older animals having humoral

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immune competence and thus higher serum globulin concentrations. Total blood protein increased with

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age in the Tasmanian devils (and decreased in the oldest devils) which is consistent with data from the

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tammar wallaby (Macropus eugenii), brushtail possum (Trichosurus vulpecula) and mountain brushtail

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possum (Trichosurus caninus), likely due to more muscle mass and higher globulin activity in older

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animals (Barnett et al., 1979a; Presidente and Correa, 1981; McKenzie et al., 2002). The decrease in total

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protein levels in 6 and 7 year old devils could be associated with senescence and living beyond their wild

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life expectancy. Generally females become senescent after about 4 years old and lifespan in the wild is

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around 5 - 6 years (Guiler, 1978; Jones, 2008).

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Total protein concentrations in Tasmanian devils were lower in autumn compared to spring and

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summer. A similar increase in total protein activity during summer has been noted in mountain brushtail

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possums (Viggers and Lindenmayer, 1996; Hufschmid et al., 2013). An increase in total protein activity is

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often associated with dehydration (Wells et al., 2000) or changes to the diet; however, as our data come

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from captive devils with constant access to water it is unlikely they were dehydrated. There was no

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simultaneous increase in HCT in the Tasmanian devils further suggesting the animals were not

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dehydrated (Barnett et al., 1979b). Changes to total protein may have been associated with changes to diet

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or associated with hormone changes in the post-breeding season.

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Seasonal changes to HGB levels have been noted in the brushtail possum, mountain brushtail

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possum, raccoon dog (Nyctereutes procyonoides), brown antechinus and tammar wallaby (Barnett et al.,

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1979a, b; McAllan et al., 1998; McKenzie et al., 2002; Mustonen et al., 2007). Generally changes in HGB

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have been associated with nutrition, but may be more associated with general body condition,

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thermoregulation or reproductive factors (Arnold, 1987; McAllan et al., 1998). Tasmanian devil HGB

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was significantly higher in spring for males which coincides with the onset of one of the bi-annual

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breeding seasons. HGB values were also higher in younger males which would be associated with the

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peak reproductive age and production of testosterone which stimulates HGB production (Shahani et al.,

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2009).

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In Tasmanian devils both season and age were found to influence AST concentrations with higher

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values observed in summer and in younger animals. Seasonal changes in AST have been observed in wild

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raccoon dogs with higher levels in autumn compared to spring (Mustonen et al., 2007). AST can be

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influenced by stress, physical examination, isolation, and can relate to liver function (McKenzie et al.,

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2002). AST has been observed to be quite high in a semelparous dasyurid, the red-tailed phascogale

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(Phascogale calura) (Bradley, 1990). Higher AST in female red-tailed phascogales was observed in June

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(winter) unlike Tasmanian devils (summer), which coincides with the month prior to breeding in each

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species (Bradley, 1990). Similar to devils, young manatees (Trichechus manatus latirostris) and young

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brush-tailed rock-wallabies have higher AST concentrations than adults (Harvey et al., 2007; Barnes et

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al., 2008).

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Gender has been shown to have a significant influence on leucocyte variables in other dasyurids,

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including the brown antechinus and red-tailed phascogale (Cheal et al. 1976; Bradley, 1990). In fat-tailed

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dunnarts the neutrophil to lymphocyte (N:L) ratio is influenced by gender (Haynes and Skidmore, 1991).

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The N:L ratio in Tasmanian devils was not found to be influenced by gender (F1,102= 0.2 P=0.63), but

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total lymphocyte and erythrocyte counts were influenced by gender with females having higher values.

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These results are confounded by small sample sizes and were only found to be significant for counts x109

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cells/L and not percentage counts for these parameters, likely due to a larger variation in percentage

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values.

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The devils included in this study were exposed to natural weather and photoperiod conditions and

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thus likely seasonal fluctuations are indicative of what would be seen in free-ranging individuals living in

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a similar climate. Gender appears to be associated with only a few variables, however some data were

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limited by a small size. More than one chemistry analyzer was used in this study and thus results may

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reflect a wider range of blood chemistry concentrations that is actually true of the devils. Statistical

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comparisons were made between the values produced from the differing biochemistry analyzers to check

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for differences. TCO2 (F1,27= 7.2 P<0.05), glucose (F1,97= 7.0 P<0.05), BUN (F1,97= 7.0 P<0.01) and

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creatinine (F1,96= 13.0, P<0.01) were the only parameters to have significantly different values between

269

the two analyzers used. These differences however would be influenced by gender, season and age of the

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animals and thus analyzers may have little impact to overall reference intervals. Further studies on

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breeding condition and lactation in females would aid future conservation efforts of the Tasmanian devil.

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This paper provides a retrospective analysis of Tasmanian devil hematology and blood chemistry

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reference intervals. The values will provide comparative data for captive animals and for those being

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translocated and released back into the wild.

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Acknowledgements

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Thank you to the staff at Taronga Zoo for collecting the data over the past 23 years. Thank you to A/Prof.

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Julie Old for providing feedback on a previous version of the manuscript. The project was approved by

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Taronga Conservation Society of Australia under license agreement R15B196. During this project HJS

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was funded as a Postdoctoral Research Associate on an Australian Research Council Linkage Grant

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(Project ID: LP140100235).

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391

17

392

Tables

393

Table 1. Hematology reference intervals, medians and confidence interval data for captive Tasmanian devils. N is

394

the number of samples analyzed and the number of animals the samples came from are in brackets. Parameter

Unit

N

Reference

Median(Range)

interval*

Erythrocytes

90% Lower

90% Upper

confidence

confidence

interval

interval

#/100WBC

19 (18)

1-20.3

3(1-23)

-

-

1012 cells/L

16 (15)

5.4-7.4

6.9(4.9-7.3)

0-6.0

7.2-7.6

MCV

fL

14 (13)

52.6-75.5

67.5(24-74)

-

-

MCH

pg

14 (13)

22.1-46.7

23.5(23-68)

-

-

MCHC

g/L

14 (13)

322.8-365.8

348(324-355)

315.1-330.8

358.2-373.9

RDW

%

14 (13)

11.23-14.97

13.5(11.8-14.8)

9.5-12.5

14.5-15.3

HGB

g/L

97 (50)

109.5-178.9

138.0(109.0-185.0)

109.0-112.5

171.6-185.0

HCT

%

102 (50)

33.7-50.0

44.0(32.0-51.0)

32.0-37.0

49.0-51.0

Platelets

HOIF1

70 (50)

5.78-37.6

15.0(5.0-38.0)

5-8

30.1-38

1012 cells/L

14 (13)

189.9-364.7

280(203-343)

155.3-223.7

329.4-396.7

WBC

109 cells/L

104 (50)

3.2-18.2

6.7(2.6-20.9)

2.6-4.0

13.0-20.9

Neutrophil

%

104 (50)

19.2-88.4

55.0(14.0-97.0)

14.0-26.7

82.5-97.0

109 cells/L

16 (15)

1.4-10.6

3.6(1.5-8.9)

1.2-1.8

7.5-13.8

%

104 (50)

7.9-79.2

42.0(0-82.0)

0-16.9

68.5-82

109 cells/L

16 (15)

0-4.6

2(0.7-4.4)

0-0.9

3.7-5.5

%

104 (50)

0-15

2(0-19.1)

0

9.4-19.1

109 cells/L

16 (15)

0-0.6

0.1(0-0.4)

0

0.4-0.9

%

104 (50)

0-4.7

0(0-8.0)

0

3.0-8.0

109 cells/L

16 (15)

-

0(0-0.2)

-

-

Lymphocyte

Monocyte

Eosinophil

395

- sample size too small for Reference Value Advisor to reliably compute confidence intervals

396

*Reference interval determined using Reference Value Advisor Excel add-on (Geffre et al., 2011)

397

1

High power oil immersion field

18

398

Table 2. Blood biochemistry reference intervals, medians and confidence interval data for captive Tasmanian devils.

399

N is the total number of samples analyzed, and in brackets are the number of animals.

400 Parameter

Unit

N

Reference

Median (Range)

interval*

90% Lower

90% Upper

confidence

confidence

interval

interval

Anion gap

mmol/L

12 (11)

8.5-19.3

13.5 (11-17)

-

-

Lipase

IU/L

6 (5)

0-151.3

60.5 (4-81)

0-13.5

91.2-216.3

GGT

IU/L

15 (14)

0-10.7

8.0 (1-10)

0-4.5

9.6-11.5

Cl

mmol/L

27 (19)

98.5-119.7

113.0 (94-119)

89.8-104.5

117.9-121.1

Total CO2

mmol/L

28 (20)

12.12-30.46

20.5 (12-30.2)

10.1-14.1

27.6-33.1

Cholesterol

mmol/L

16 (8)

2.1-6.7

3.7 (2.3-6.1)

1.7-2.6

5.6-7.9

CK

IU/L

71 (38)

220.2-2055.8

400 (201-2303)

201-243.4

1283-2303

Glucose

mmol/L

98 (48)

2.8-6.7

5.2 (2.7-7.0)

2.7-3.4

6.4-7.0

BUN

mmol/L

98 (48)

8.7-19.7

12.7 (7.2-23.8)

7.2-9.6

16.9-23.8

Creatinine

umol/L

98 (48)

29.6-107.9

61.5 (25-114)

25-33.9

93.3-114

Ca2+

mmol/L

64 (39)

2.1-2.9

2.3 (2.1-3)

2.1-2.2

2.7-3

P

mmol/L

94 (48)

0.9-3.3

1.7 (0.7-3.7)

0.7-1.1

2.8-3.7

Na+

mmol/L

57 (28)

133.8-148.0

142 (132-148)

132-139

148

K+

mmol/L

60 (28)

3.6-6.3

4.5 (3.6-6.8)

3.6-3.8

5.4-6.8

Mg2+

mmol/L

15 (14)

0.5-1.0

0.7 (0.5-1.0)

0.5-0.6

0.9-1.5

Total Protein

g/L

92 (47)

49.3-76.0

63.5 (49-78)

49-51.3

72.7-78

Globulin

g/L

92 (47)

5.3-51.0

25 (4-60)

4-7.6

50-60

Albumin

g/L

93 (47)

16.0-52.7

39 (15-54)

15-18

49-54

ALT

IU/L

97 (48)

15.6-43.0

25 (13-54)

13-16.9

37-54

Total bilirubin

umol/L

52 (33)

1.3-9.0

5.2 (1-9)

1-2

7-9

AMY

IU/L

35 (26)

62.3-511.2

102 (69-371)

58.6-66.6

251.9-1473.2

ALP

IU/L

94 (46)

49.3-279.1

112.5 (49-281)

49-62

238-281

19

AST

401

IU/L

67 (36)

16.1-104.3

44 (18-111)

18-20.7

96-111

*Reference interval determined using Reference Value Advisor Excel add-on (Geffre et al., 2011)

402

20

Table 3. Hematology and serum biochemistry (mean ± SD) variables of captive Tasmanian devils that were significantly influenced by their age. Other variables did not differ significantly between age classes. Age (years)

Under 1

1

2

3

4

5

6

7

Number of samples

12

28

11

25

12

2

8

6

WBC (109 cells/L)

7.8 ± 2.3d

9.9 ± 4.4b

6.9 ± 1.5

6.4 ± 2.1a

6.2 ± 2.6a,c

6.4 ± 2.7

6.5 ± 1.6

7.2 ± 1.5

HCT (%)

42.3 ± 1.2

45.1 ± 2.4a

45.0 ± 3.3a

45.8 ± 3.6a

46.9 ± 2.3a

41.5 ± 0.7

41.0 ± 5.1

37.7 ± 4.6b

Neutrophils (%)

43.3 ± 9.4b

46.0 ± 16.1b

63.0 ± 17.6a

53.8 ± 13.0c

57.4 ± 14.1

56.4 ± 3.6

71.8 ± 8.5a

77.2 ± 7.1a,d

Lymphocytes (%)

54.5 ± 10.2a

50.0 ± 16.6a

33.7 ± 18.3

41.9 ± 12.4a

38.4 ± 14.4

33.1 ± 2.8

26.6 ± 8.4b

20.3 ± 7.7b

Number of samples

7

25

11

22

13

2

6

8

Globulin (g/L)

10.9 ± 8.6g

23.1 ± 10.1

27.8 ± 10.3

32.9 ± 14.0

27.9 ± 11.2

21.0 ± 1.4

25.2 ± 1.1

26.9 ± 7.2

Total protein (g/L)

52.9 ± 3.7c

62.0 ± 6.3d

65.2 ± 5.7a,d

66.8 ± 5.9a,d

65.7 ± 4.5a,d

61.2 ± 3.9

56.9 ± 4.1

56.9 ± 4.1b

ALP (IU/L)

236.7 ± 32.2a

178.0 ± 47.9a

124.9 ± 39.2b

96.0 ±31.2b

91.5 ± 24.3b

95.5 ± 2.1b

84.7 ± 4.1b

79.5 ± 16.6b

AST (IU/L)

76.7 ± 23.3a

40.7 ± 25.6b,c

50.6 ± 15.7

37.2 ±13.3b

43.8 ± 14.7

20.0 (n=1)

51.2 ± 21.3

71.3 ± 23.2d

P (mmol/L)

2.9 ± 0.6e

1.8 ± 0.4

1.7 ± 0.3

1.7 ± 0.4

1.4 ± 0.3

1.5 ± 0.3

1.9 ± 0.4

1.6 ± 0.5

a-b, c-d

e

significantly different from each other P<0.01

significantly different from all other values P<0.01

21

Table 4. Hematology and serum biochemistry (mean ± SD (n)) variables of captive Tasmanian devils that were significantly influenced by gender and season. Parameter

Unit

Gender Male

Erythrocytes

Season Female

Summer

Autumn

Winter

Spring

#/100WBC

2.6 ± 1.4 (11)

5.6 ± 7.1 (8)

4.6 ± 6.2 (11)

2.5 ± 0.7 (2)

3.0 (2)

3.0 ± 1.6 (5)

1012 cells/L

6.4 ± 0.7a (10)

7.1 ± 0.1 (6)b

6.9 ± 0.3 (10)

6.9 (1)

4.9 (1)

6.5 ± 0.6 (4)

HGB

g/L

140.8 ± 18.2 (56)

141.6 ± 17.5 (41)

138.3 ± 15.3 (45)a

139.3 ± 19.0 (20)a

129.2 ± 10.7 (5)

149.6 ± 19.3 (27)b

Lymphocyte

%

40.2 ± 17.5 (60)

43.7 ± 15.0 (44)

45.0 ± 17.0 (49)

39.2 ± 17.4 (20)

34.8 ± 15.5 (5)

39.0 ± 14.9(30)

109 cells/L

1.8 ±0.8a (9)

2.8 ± 1.1 (7)b

2.4 ± 1.1 (9)

1.2 (1)

0.7 (1)

2.4 ± 0.8(5)

Lipase

IU/L

14.5 ± 14.8 (2)a

70.5 ± 16.5 (4)b

35.5 ± 14.8 (2)

60.0 ± 37.4 (4)

- (0)

- (0)

Total

g/L

62.1 ± 6.6 (50)

63.4 ± 6.6 (42)

63.4 ± 7.4 (38)a

58.1 ± 5.3 (18)b

62.2 ± 6.2 (6)

64.7 ± 5.2 (30)a

IU/L

120.5 ± 76.8 (22)a

187.2 ± 102.5

149.5 ± 102.0 (15)

182.3 ± 107.8 (10)

77.0 (1)

104.6 ± 17.8 (9)

54.6 ± 25.3 (29)c

51.9 ± 21.8 (14)

55.4 ± 29.1 (5)

34.5 ± 14.1 (19)d

Protein AMY

(13)b AST a-b, c-d

IU/L

50.6 ± 25.2 (36)a

45.7 ± 21.3 (31)b

Statistically different from one another P<0.05

22